Fermentation process

ABSTRACT

A method for improving the yield of 7-(D-5-amino-5carboxylvaleramido)-3-(carbamoyloxymethyl)-7-methoxy-3-cephem -4carboxylic acid by adding to fermentation media an additive selected from glycine, L-phenylalanine, a carbamate or an amide, or combinations thereof. The product is a new antibiotic produced from a new strain of Streptomyces; it exhibits activity against gram-negative and gram-positive bacteria.

United States Patent [19'] Birnbaum et al.

in] 3,769,169 Oct. 30, 1973 FERMENTATION PROCESS Inventors: JeromeBirnbaum, Morganville;

Edward Inamine, Rahway, both of NJ.

Assignee: Merck & Co., Inc., Rahway, NJ.

Filed: May 20, 1971 Appl. No.: 145,573

US. Cl 195/80, l95/36, 260/243 C Int. Cl. Cl2k 1/00 Field of Search195/36 R, 80

References Cited UNITED STATES PATENTS 4/1969 Arnold et a1 195/36 RPrimary Examiner-A. Louis Monacell Assistant Examiner-Robe'rt J. Warden1 t ttorney Rudolph J. Anderson, J r. et al.

ABSTRACT 12 Claims, No Drawings FERMENTATION PROCESS This inventionrelates to an improved fermentation process for the production of theantibiotic 7-(D-5- amino-5-carboxyvaleramido)-3-(carbamoyloxymethyl)-7-methoxy-3-cephem-4- carboxylic acid. Inparticular, this invention relates to an improved method for theproduction of the. said antibiotic via the fermentation of nutrientmedia with suitable strains of the microorganism Streptomyceslactamdurans.

The Streptomyces Iaetamdurans culture is a new strain of actinomyceteand a sample of this microorganism, designated as MA-2908, has beenplaced in the culture collection of Merck & Co., lnc., Rahway, NewJersey. A sample of this culture has also been placed on permanentdeposit with the culture collection of the Northern Utilization Researchand Development Branch of the US. Department of Agriculture at Peoria,Illinois. This culture has been assigned the culture number NRRL 3802.

The 7-(D-5-amino-5-carboxyvaleramido)-3-(carbamoyloxy-methyl)-7-methoxy-3-cephem-4- carboxylic acid (hereinafterreferred to as the antibiotic) is produced during the aerobicfermentation of suitable aqueous nutrient media under controlledconditions. Aqueous media such as those employed for the production ofother antibiotics are suitable. Such media contain sources of carbon andnitrogen which are assimilable by the microorganism and inorganic salts.In addition, the fermentation media contain traces of metals necessaryfor the growth of the microorganism which are commonly supplied asimpurities incidental to the other constituents of the medium. Ingeneral, carbohydrates such as sugars, for example, sucrose, maltose,fructose, lactose and the like, and

starches such as grains, for example oats and rye, corn starch, cornmeal and the like, can be used either alone or in combination as sourcesof assimilable carbon in the nutrient medium. The exact amount of thecarbohydrate source or sources utilized in the medium will depend inpart upon the other ingredients of the medium. It has been found,however, that an amount of carbohydrate between about-l and .6 percentby weight of the medium is sufficient. A single carbon source may beused, or several carbon sources may be combined in the medium.

Satisfactory nitrogen sources include myriad proteinaceous materialssuch as various forms of hydrolysates of casein, soybean meal, cornsteep liquor, distilled sol-.

ubles, yeast products tomato paste, and the like. The various sources ofnitrogen can be used either alone or in combination, and are used inamounts ranging from 0.2 6 percent by weight of the aqueous medium.

The expression: complex organic media as used in this specificationmeans media wherein some of the ingre-dients are not chemically defined.An example of I 2 7-(D-5-Amino-5-carboxyvaleramido)-3-(carbamoyloxymethyl)-7-methoxy-3-cephem-4- carboxylic acid has thefollowing planar formula:

0 NH: O OCH: S nolLhH-cm-om-om-l J-Nrri f1} O 4 3 cmoliNm OOH Thiscompound (l) is produced during the aerobic fermentation described abovefrom a strain of Streptomyces lactamdurans capable of producing saidcompound as, for example, from the strain on permanent deposit at theNorthern Utilization Research and Development Branch under accessionnumber NRRL 3802. Other strains of this species, such as mutantsobtained by mutating agents or isolated from nature, can also be used.The antibiotic (I) is amphoteric' with an apparent isoelectric point-ofabout pH 3.5 and is stable in solution at a pH range of about 1.5 9.0.

The antibiotic and its salts demonstrate resistance not only topenicillinase but to the cephalosporinases as well and exhibit anenhanced activity against gramnegative microorganisms. Unlike'cephalosporin C which has a relatively low antibacterial activity, theantibiotic of this invention and its salts exhibit a significant in vivogram-negative effect with a potency which, in general, is greater thancephalothin. This activity includes effectiveness in vivo on Proteusmorganii and, in addition, an effectiveness against the followinggramnegative bacteria: Escherichia coli, Proteus vulgaris, Proteusmirabilis, Salmonella schottmuelleri, Klebsiella pneumoniae AD,Klebsiella pneumbniae B and Paracolobactrum arizoniae.

Bioassays for this antibiotic are run by a disc-plate procedure usinginch filter paper discs. The assay plates are prepared using Difconutrientagar plus 2.0 g./l. Difco yeast extract at 10 ml. per plate. Anovernight growth of the assay organism, Vibrio percolans MB-l272 isdilutedin sterile salinesolution to a suspension having 40 percenttransmittance at a wave length of 660 my" Thissuspension is added to 20ml. /liter to medium prior to pouring the plates. The Vibrio percolansorganism is on deposit in the Culture Collection of the American TypeCulture Collection where it is available under the followingdesignation: Vibrio percolans MB-l272, ATCC 8461.

The assay plates are held at 4C. until used (5 day maximum). Followingthe application of the antibioticsaturated assay discs the plates areincubated at 28C. for a period of from 8 to 24 hours. Zones ofinhibition areread as mm. diameter. They are used to determine relativepotencies or, when compared with a purified reference standard, thepotency in pug/ml.

Due to the inherent difficulty in separating pure 7- (D5-amino-5-carboxyvaleramido)-3-(carbamoyloxymethyl)-7-methoxy-3-cephem-4- carboxylic acid from thelarge quantities of impurities in the fermentation broth, itis of greatimportance to find a way to increase the concentration of the antibioticrelative to the total broth solids.

It is, therefore, an object of this invention to provide a method ofincreasing the yield of the antibiotic in a wherein R is alkyl, forexample, lower alkyl such as ethyl, n-propyl or n-butyl and the like andR and R are the same or different members selected from hydrogen, alkyl,for example, lower alkyl such as methyl, ethyl, n-propyl or n-butyl andthe like or hydroxy-lower alkyl, for example, 2-hydroxyethyl or3-hydroxypropyl and the like; R is hydrogen, alkyl, such as methyl,ethyl, npropyl, n-butyl, n-pentyl or undecyl and the like, oxosubstituted lower alkyl such as 2-oxopropyl and the like or aryl, forexample, mononuclear aryl such as phenyl; and R and R are the same ordifferent members selected from hydrogen, alkyl, for example, loweralkyl such as methyl, ethyl, n-propyl, n-butyl, isobutyl or npentyl andthe like, or hydroxy-lower alkyl such as 2- hydroxyethyl or3-hydroxypropyl and the like; and X is oxygen or sulfur.

The amount of glycine, L-phenylalanine, carbamate (ll) or amide (ill)needed to stimulate production of the antibiotic varies with the mediumemployed. In general, increased production of the antibiotic has beenobserved in media containing from 0.01 0.10

percent (weight/volume) of glycine and from 0105 0.3

percent (weight/volume) of L-phenylalanine. However, preferredconcentrations are 0.05% glycine and 0.3% L-phenyl-alanine. Thecarbamates (II) and amides (III) are employed in the range of from about0.0156 2.0 percent and especially good yields have been obtained withN-Z-hydroxyethyl alkylcarbamates as, for example, with from about 0.20.8 percent of ethyl N-2-hydroxyethylcarbamate.

Also, we have found that the addition of an amide of the followingformula to the fermentation medium will effect a significant increase inyield of antibiotic:

0 /R1 Ril lN\' wherein R,, is hydrogen or lower alkyl such as methyl orethyl and R and R are hydrogen or lower alkyl especially isobutyl. Thecompound N,N- diisobutylpropionamide is particularly preferred andaffords superior yields when employed at a concentration of from about0.063 0.125 of the medium employed. Concentrations of glycine greaterthan 0.3 percent, concentrations of L-phenylalanine greater than 0.5percent and concentrations of carbamates greater than 0.8 percent tendto decrease production of the antibiotic.

In addition to being used singly we have found that glycine,L-phenylalanine, and a carbamate of Formula ll, supra, and/or an amideof Formula lIl, supra, may be combined to afford an additive which willinduce a particularly good yield of antibiotic. Thus, for example, thecombination of 0.05 glycine, 0.3% L- phenylalanine and an amount ofcarbamate (ll) and/or amide (ill) falling within the range of 0.0156 2.0percent final concentration is particularly suitable in stimulating theyield of antibiotic. Preferably, in this combination, the carbamate isethyl N-2-hydroxyethylcarbamate at a final concentration of 0.2 0.8percent and the amide is N ,N-diisobutylpropionamide at a finalconcentration of 0.063 0.125 percent.

The above discussion is primarily directed to fermentations using theparent strain Streptomyces lactamdurans. However, other strains of thisorganism such as mutants, can also be used to produce the antibiotic. It

- is deemed obvious to one skilled in the art that thecarcarboxyvaleramido )-3-(carbamoyloxymethyl )-7-methoxy-3-cephem-4-carboxylic acid (1).

Although the antibiotic (I) of this invention is produced by bothsurface and submerged cultures, it is presently preferred to carry outthe fermentation in the submerged state. Small scale-fermentations areconveniently carried out by placing suitable quantities of nutrientmedium in flasks, sterilizing the flasks and contents by heating tol20C., inoculating the flasks with either spores or a vegetativecellular growth of a 7-(D- S-amino-S-carboxyvaleramido)-3-(carbamoyloxymethyl)-7-methoxy-3-cephem-4- carboxylic acid producingstrain of Streptomyces, loosely st'oppe'ring the necks of the flaskswith cotton, and permitting the fermentation to proceed in a constantroom temperature at about 28C. on a shaker for 35 days. For larger scalework, it is preferable to conduct the fermentation in suitable tanksprovided with an agitator and a means of aerating the fermentationmedium. In this method, the nutrient medium is made up in the tank andsterilized by heating at l20C. After cooling, the sterilized medium isinoculated with a suitable source of vegetative cellular growth of theStreptomyces and the fermentation is permitted to proceed for 2-4 dayswhile agitating and/or aerating the nutrient medium and maintaining thetemperature at about 28C. This method of producing 7-(D-5-amino-5-carboxyvaleramido)-3-(carbamoyloxymethyl)-7-methoxy-3-cephem-4-carboxylic acid is particularly.

suited for the preparation of large quantities of the new antibiotic.

The fermentation using the7-(D-5-amino-5-carboxyvaleramido)-3-(carbamoyloxymethyl)-7-methoxy-3-cephem-4-carboxylic acid producing microorganism can be carried out attemperatures ranging from about 20379C. For optimum results, however, itis most convenient to conduct the fermentations at temperatures between26-30C, The pH of the nutrient media suitable for growing theStreptomyces and producing the antibiotic may vary from about 5 to 9.The preferred pH range, however, is from about 6.0 to 7.5.

In carrying out the invention, a cell suspension is prepared by theaddition of sterile medium to an agar slant culture of a7-(D-5-amino-S-carboxyvaleramido)-3-(carbamoyloxymethyl)-7-methoxy-3-cephem-4- carboxylic acid producingmicroorganism. Growth from the slant culture is then used to inoculate aseed flask and the seek fl'ask is shaken at about 28C. for 1-3 days inorder to obtain good growth. The seed flask is then used to inoculatethe production flasks. Alternatively, the seed flask can be inoculatedfrom a lyophilized culture or a frozen inoculum.

The inoculation is generally carried out using about 1 ml. per 40 ml. ofproduction medium. The desired concentration of additive is then addedto the production flasks and the fermentation is permitted to proceedfor 2-4 days while agitating and/or aerating the nutrient medium andmaintaining the temperature at about 28C. All of the production flasks,i.e., those containing additives and the flisks used as controls, arethen assayed, generally after 96 hours, to determine the amount ofantibiotic produced in each flask.

The antibiotic 7-(D-5-amino-5-carboxyvaleramido)-3-(carbamoyloxymethyl)-7-methoxy-3-cephem-4- carboxylic acid wereconveniently assayed by means of a disc-plate procedure using Vibriopercolans MB-l272 (ATCC 8461) as the assay organism. Discs of ifs-inchdiameter were used. A standard curve was prepared from knownconcentrations of the antibiotic and activity was expressed inmicrograms, i.e., mg., per milliliter of free acid.

The production flasks were then assayed by diluting the sample in 0.02molar phosphate buffer at pH 7 to an appropriate concentration. The testorganism was Vibrio percolans MB-l272 (ATCC 8461 and the assay mediumemployed was Difco nutrient agar plus 0.2% Difco yeast extract. Thediscs were dipped into 5 mg. per milliliter of the standard antibioticsolution, and were placed on the plate in a position alternate to thesample. The plates were then incubated at 37C. for 18 hours, and thezone diameters in millimeters were determined. Five standard platescontaining four levels of the standard ranging from 2.5 to 20 mg./rnl.were employed. The assay was calculated by means ofa Nomograph and theresults reported in terms of micrograms per milliliter.

The antibiotic can be recovered from the fermentation medium by a numberof procedures. The filtered broth can be passed through one or more ionexchange columns. The amphoteric nature of the antibiotic (I) enablesselection of both cationic and anionic ion exchange resins to optimizerecovery. The adsorbed antibiotic can then be removed by elution,preferably in a volatile solvent such as pyridine which can be easilyremoved.

The antibiotic 7-(D-5-amino-5-carboxyvaleramido)-3-(carbamoyloxymethyl)-7-methoxy-3-cephern-4- carboxylic acid (I) andits salts are efiective in inhibiting the growth of variousgram-negative and grampositive microorganisms. I

The following examples are given for purposes of illustration and not byway of limitation.

EXAMPLE 1 culture (MA-2908) was opened asceptically and the organismtransferred to a medium of the following composition:

Medium 1:

1 percent Blackstrap Molasses 1 percent National Brewers Yeast 2.5percent Difco Agar pH 7.0

Water to Volume The slants are incubated for seven days at 28C. Whenstored in the cold, the slants are stable for more than 13 weeks.

Step B: Seed Stages: Two Stage System First Seed: The first seed isinoculated directly from the slant of Step A to 40 ml. of 1 percentPrimary Dried Yeast N.F., pH 7.0 (obtained from the Yeast ProductCorporation) in a 250 ml. baffled Erlenmeyer flask. The flasks were thenshaken on a 220 rpm. rotary shaker with a two. inch-throw at 28C. for aperiod of from two to three days.

Second Seed: A 2.5 percent inoculum from the first seed stage was addedto a flask containing a 2% Fleischmann S-l50 yeast autolysate, pH 7.0.The growth in this stage is characteristically light and the incubation,performed as in the first stage at 28C, was not extended beyond 48hours.

Step C: BasalProduction Medium The basal production. medium had thefollowing composition:

Medium ll:

Distillers Solubles 3.0%

Primary Dried Yeast 0.75%

Mobil par-S Defoamer 0.25%

This medium was adjusted to pH 7.0 with a small amount of concentratedsodium hydroxide solution, dispensed into Erlenmeyer flasks andautoclaved for 15 to 20 minutes at 121C. After cooling the mediumreceived a 2.5 percent inoculum of the second stage seed obtained inStep B. lncubation'was for three days at 28C. on a 220 rpm rotary shakerwith a two inch throw.

When the fermentation was complete, the cells were removed bycentrifugation and the broth was diluted with phosphate buffer, pH 7.0.The concentration of 7-(D-5-amino-5-carboxyvaleramido)-3-(carbamoyloxymethyl)-7-methoxy-3-cephem-4- carboxylic acid in thefermentation broth was determined by the standard biological-disc assaymethod.

The assay organism employed was Vibrio percolans (ATCC 8461 Filter paperdiscs were emersed in the diluted broths and placed on the surface ofagarcontaining Petri dishes that had been inoculated with the assayorganism Vibrio percolans (ATCC 8461). Also placed on these Petri disheswere discs that had been dipped previously in standard solutionscontain-' ing known concentrations of 842A. The discs were incubatedovernight at 28C. and the diameters of the zones of inhibition recorded.The concentration of 842A and the fermented broth is calculated byinterpolation from the standard curve which relates zone diameter withthe known concentrations of standard 842A solutions. By this procedureit was calculated that Streptomyces lactama'urans MB-2908 produced anaverage of 80.4 ug/ml. of 7-(D-5-amin0-5- carboxyvaleramido)-3-(carbarnoyloxymethyl )-7- methoxy-3-cephem-4-carboxylic acid in thebasal production medium.

Step D: Addition of Glycine The addition of glycine to Medium 11 in StepC, supra, increased the yield of 7-(D-5-amino-5-carboxyvaler-amido)-3-(carbamoyloxymethyl)-7-methoxy-3-cephem-4-carboxylic acid significantly. Table l belowindicates the extent of this increase over a range of concentrations.The data in this Table represents three separate series of tests inwhich the control was conducted in the same manner as described in StepsA-C above and in which the remaining experiments were conducted in anidentical manner but with the addition of the indicated amount ofglycine. The assays were conducted in the same manner as in Step C.

TABLE I 71 Glycine Antibiotic Experi' Added to Production lncrease mentMedium ll (ug/ml) Over Control Control 77 2 0.10 97 26% Control 76 20.10 93 22% Control 88 On the basis of the foregoing the addition of0.05% glycine to the production medium was judged most effective inincreasing the yield of 7-(D-5-amino-5-carboxyvaleramido)-3-(carbamoyloxymethy1)-7-methoxy-3-cephem-4-carboxylic acid.

EXAMPLE 2 Antibiotic Production; Addition of L-Phenylalanine Theprocedure of Example 1 was repeated except that various concentrationsof L-phenylalanine were substituted for the glycine additive in Step D.The following Table indicates the concentrations at which L- phenylanine'was employed and the increase in yield of Antibiotic (l) attributedthereto.

TABLE ll 17 Experi- /r L-Phcnylalanine Antibiotic Increase Over mentadded to Medium ll Production (pug/ml) Control Control 76 l 0.05 82 8% 20.1 77 1'7? 3 0.3 90 34% Control 85 EXAMPLE 3 Glycine 0.05% Thefollowing Table indicates the concentrations at which L-phenylalaninewas employed and the increase in yield of Antibiotic (l) attributedthereto.

TABLE 111 Antibiotic Experi- L-Phenylalanine Production Increase mentadded to Medium lll (ug/ml) Over Control Control 101 l 0.1 124 23%Control 103 l 0.3 1 18 15% Control 1 l0 EXAMPLE 4 Antibiotic Production;Addition of Ethyl Carbamate and Glycine The procedure of Example 3 wasrepeated except that ethyl carbamate was substituted for L-phenylalanine. The production medium employed was Medium ill of Example3 which contains 0.05% glycine. The following Table indicates theconcentrations at which ethyl carbamate was employed and the increase inyield of Antibiotic (l) attributed thereto.

Each yield is the average of 8 individual assays. On the basis of theforegoing the addition of0.4% ethyl carbamate to Medium 111 was mosteffective in increasing the yield of Antibiotic (1).

EXAMPLE 5 Antibiotic Production; Addition of Amides The procedure ofExample 1, Steps A-C was repeated except that the incubation period ofStep C was for four days at 28C. and the basal production medium had thefollowing composition:

Medium 1V:

Distillers Solubles 3.0%

Primary Dried Yeast 1.0%

Glycine 0.05%

L-Phenylalanine 0.3%

Mobile par S-Defoamer 0.25%

Sodium Thiosulfate 0.1%

Cornstarch 2.0% Upon adding the amides in the following Table to Medium1V and otherwise following the procedure described in Example 1, Step C,an increase in the yield of Antiobiotic (l) was noted. Table V infradescribes these amides, the concentrations at which they were employedand the increases in yield attributed thereto.

TABLE V Antibiotic Production (ptg/ml) Final with Additive/ControlAdditive Concentration No. 1 No. 2 Formamide 0.25 332/256 0.50 354/256NMethyl- 1.0 285/254 formamide 2.0 312/254 N.N-Diethyl- 0.25 287/238formamide 0.40 346/273 301/256 0.50 297/238 0.80 381/273 328/256 1.0388/273 387/256 N.N,Diethyl- 0.25 309/254 formnmide 0.5 351/254 1.0391/254 N,N.Dibutyl 0.125 260/254 formamide 0.25 285/254 N-Z-Hydroxy-0.25 310/256 ethylformaide 0.50 338/256 7 1.0 372/256 N,N-Dimcthyl- 0312293/256 benzamide 0.0625 313/256 0.125 369/256 N,N-Dimethyl- 0.5 360/254acetamide 1.0 354/254 2.0 449/254 N,N-Diethyl- 0.25 276/238 acetamide0.5 379/238 10 335/238 N,N-Dipropyl- 0.16 340/238 acetamide 0.31 390/238N.N-Dimethyl- 0.063 296/256 thiuacetamide 0.125 288/254 330/256 0.25145/254 322/256 N.N-Dimethyl- 0.5 293/254 ucetoucetamide 1.0 286/254 2.0380/254 N.N-Dimethylpro- 0.5 363/254 pionamide 1.0 369/254 2.0 282/254N.N-Dibutylpro- 0.0156 7 418/256 pionumide 0.0312 528/256 0.0625 350/256N.N-Diisobuty1- 0063 497/256 pripionumide 0.125 530/256 N,N-Dimethyl-0.125 32-2/254 hutyramide 0.25 303/254 0.5 318/254 N.N-Dimethyl- 0.063308/254 valerumide 0.125 325/254 0.25 355/254 N.N-Dimethy1dn- 0.0156291/256 decunumide EXAMPLE 6 Antibiotic Productioh; Addition ofCarbamates The procedure of Example 5 was repeated except that variouscarbamates were substituted for the amides recited therein. Thefollowing table describes these carbamates and indicates theconcentrations at which they were employed and the yield of antibiotic(l) attributed thereto:

TABLE VI Antibiotic Production (ug/ml) thylcarbam ate What is claimedis:

l. 1n the method for preparing 7-( D-5-amino-5-carboxyvaleramido)-3-(carbamoyloxymethyl)-7-methoxy-3-cephem-4-carboxylic acid by growing Streptomyces lactamduransor a mutant thereof in a nutrient medium; the improvement whichcomprises adding a member selected from the group consisting of glycine,L-phenylalanine, a carbamate, an amide or combinations thereof to thefermentation medium.

2. The method of claim 1 wherein the pH of the aqueous nutrient mediumis in the range of from about 6.0 to 8.0 and contains between about 1percent and 6 percent by weight of carbohydrate and between about 0.2percent and 6 percent by weight of available nitrogen.

3. The method of claim 1 wherein the carbamate is a compound of theformula:

whereinR is alkyl and R and R are the same or different members selectedfrom hydrogen, alkyl or hydroxylower alkyl; and the amide is a compoundof the formula:

wherein R is a-hydrogen, alkyl, oxo substituted lower alkyl or aryl andR and R are the same or different members selected. from hydrogen,alkyl, or hydroxylower alkyl.

4. The method of claim 3 wherein the amide is formamide or N,N-di-1oweralkyl formamide.

5. The method of claim 3 wherein the amide employed isN,N-diisobutylpropionamide at a concentration of from about 0.063 0.125percent.

6. The method of claim 3 wherein the carbamate employed isN-Z-hydroxyethylcarbamate at a concentration of from-about'0.2 0.8percent.

7. The method of claim 3 wherein the carbamate or amide is employed at aconcentration of from about 0.0156 2.0 percent.

8. The method of claim 3 wherein the amide is N,N-dimethylformamide.

9. The method of claim 1 wherein glycine is employed at a concentrationof from about 0.01 0.10

percent.

10. The method of claim 1 wherein L-phenylalanine is employed at aconcentration of from about 0.05 0.3

percent.

11. In the method for preparing 7-(D-5-amino-5-carboxyvaleramido)-3-(carbamoyloxymethyl)-7-methoxy-3-cephem-4-carboxylic acid by growing Streptomyces lactamduransin a nutrient medium, the

improvement which comprises using 0.05% glycine, 0.3% L-phenylalanine,0.2 0.8 percent ethyl N-Z-hydroxy-ethylcarbamate or 0.063 0.125 percentN,N-diisobutylpropionamide as an additive to the fermentation medium toimprove the yield of antibiotic.

12. The method of claim 11 wherein either N-2-hydroxyethylcarbamate orN,N- diisobutylpropionamide is used in combination with glycine andL-phenylalanine.

UNITED STATES PATENT eTTTcE CERTIFICATE 6F CQRREQTWN Patent No. ,169Dated October 30, 1973 Inventor) Jerome Birnbaum and Edward Inamine Itis certified that error appears in the above-identified patent and thatsaid Letters Patent are hereby corrected as shown below:

In Column 9, Table V, the name of the third additive should correctly be"N,NDimethylformamide" Signed and sealed this 2nd day of April 1.97L

(SEAL) Attest: I

EDWARD M.FLETCHER,JR. C. MARSHALL DANN Attesting Officer Commissioner ofPatents

2. The method of claim 1 wherein the pH of the aqueous nutrient mediumis in the range of from about 6.0 to 8.0 and contains between about 1percent and 6 percent by weight of carbohydrate and between about 0.2percent and 6 percent by weight of available nitrogen.
 3. The method ofclaim 1 wherein the carbamate is a compound of the formula:
 4. Themethod of claim 3 wherein the amide is formamide or N,N-di-lower alkylformamide.
 5. The method of claim 3 wherein the amide employed isN,N-diisobutylpropionamide at a concentration of from about 0.063 -0.125percent.
 6. The method of claim 3 wherein the carbamate employed isN-2-hydroxyethylcarbamate at a concentration of from about 0.2 - 0.8percent.
 7. The method of claim 3 wherein the carbamate or amide isemployed at a concentration of from about 0.0156 - 2.0 percent.
 8. Themethod of claim 3 wherein the amide is N,N-dimethylformamide.
 9. Themethod of claim 1 wherein glycine is employed at a concentration of fromabout 0.01 - 0.10 percent.
 10. The method of claim 1 whereinL-phenylalanine is employed at a concentration of from about 0.05 - 0.3percent.
 11. In the method for preparing7-(D-5-amino-5-carboxyvaleramido)-3-(carbamoyloxymethyl)-7-methoxy-3-cephem-4-carboxylic acid by growing Streptomyces lactamdurans in a nutrientmedium, the improvement which comprises using 0.05% glycine, 0.3%L-phenylalanine, 0.2 - 0.8 percent ethyl N-2-hydroxy-ethylcarbamate or0.063 - 0.125 percent N,N-diisobutylpropionamide as an additive to thefermentation medium to improve the yield of antibiotic.
 12. The methodof claim 11 wherein either N-2-hydroxyethylcarbamate orN,N-diisobutylpropionamide is used in combination with glycine andL-phenylalanine.